Final Report Conservation Genetics of Spring Associated Darters in Alabama Prepared by: Brook L. Fluker, Bernard R. Kuhajda, and Phillip M. Harris Department of Biological Sciences University of Alabama Box 870345 Tuscaloosa, Alabama 35487-0345 Submitted to: Alabama Department of Conservation and Natural Resources State Wildlife Grants Program 30 December 2011 Contents Project Overview .............................................................................................................................1 References ........................................................................................................................................4 1. Evolutionary History and Conservation Genetics of the Slackwater Darter (Etheostoma boschungi) and Tuscumbia Darter (Etheostoma tuscumbia) .......................................................6 Introduction ..........................................................................................................................6 Materials and Methods .........................................................................................................9 Results ................................................................................................................................17 Discussion ..........................................................................................................................22 Conservation implications and recommendations .............................................................28 References ..........................................................................................................................39 Tables .................................................................................................................................46 Figures................................................................................................................................52 2. Evolutionary History of the Etheostoma swaini Species complex in the Mobile Basin, with Emphasis on Conservation Genetics of the Coldwater Darter (E. ditrema) ..............................56 Introduction ........................................................................................................................56 Materials and Methods .......................................................................................................58 Results ................................................................................................................................65 Discussion ..........................................................................................................................69 Conservation implications and recommendations .............................................................73 References ..........................................................................................................................83 Tables .................................................................................................................................87 Figures................................................................................................................................93 3. Conservation Genetics of the Rush Darter (Etheostoma phytophilum): Implications for Conservation of Spring Endemic Fishes of the Southeastern United States ..............................97 Introduction ........................................................................................................................97 Materials and Methods .....................................................................................................101 Results ..............................................................................................................................108 Discussion ........................................................................................................................113 Conservation guidelines for spring endemic species and recommendations for Etheostoma phytophilum ..................................................................................................118 References ........................................................................................................................126 Tables ...............................................................................................................................135 Figures..............................................................................................................................144 ii Project Overview Organisms inhabiting freshwater springs present unique challenges to conservationists and natural resource managers. The naturally fragmented distribution of spring species render them extremely vulnerable to fine-scale disturbance and springs are among the most anthropogenically exploited freshwater habitats (Meffe and Vrijenhoek 1988; Hubbs 1995; Etnier 1997; Timpe et al. 2009; Fluker et al. 2010; Martin 2010). Spring endemic species are often confined to spring pools and short stretches of spring runs, with interconnecting streams and rivers acting as major barriers to dispersal (Starnes and Etnier 1986). Consequently, spring endemic species should share several characteristics with island endemic species, i.e. small genetically structured populations with low genetic diversity, making them more susceptible to local extinction compared to their mainland or widespread relatives (Frankham 1997; Wilson et al. 2009). Recent studies of spring taxa support this hypothesis in terms of genetic structure (Martin and Wilcox 2004; Hurt and Hedrick 2004; Wilmer and Wilcox 2007) and small population sizes with low genetic diversity (Duvernell and Turner 1999; Fluker et al. 2010). Most genetic studies of North American spring endemic species have focused on taxa from arid lands (Vrijenhoek et al. 1985; Meffe and Vrijenhoek 1988; Echelle et al. 1989; Thompson et al. 2002; Martin and Wilcox 2004; Hurt and Hedrick 2004; Bernardi et al. 2007) where demands from municipal and agricultural users for groundwater have long conflicted with biodiversity conservation (Deacon et al. 2007). Relatively few genetic studies have been conducted in the southeastern United States, however, where recent groundwater demands due to rapid human population growth threaten the rich diversity of coldwater spring endemics (Hubbs 1995; Etnier 1997; Mirarchi et al. 2004). 1 Within the state of Alabama, seven darter species (Percidae: Etheostoma) either permanently inhabit springs or require springs and spring seeps for reproduction. The Watercress Darter (Etheostoma nuchale) and Tuscumbia Darter (E. tuscumbia) are permanent residents of springs and their associated spring runs. The Coldwater Darter (E. ditrema), Goldstripe Darter (E. parvipinne), and Rush Darter (E. phytophilum) inhabit small headwater streams, springs, spring runs, and seeps. In contrast, the Slackwater Darter (E. boschungi) and Trispot Darter (E. trisella) normally occupy small to moderately large streams, but migrate into ephemeral seeps during winter months for reproduction. These spring associated darters are some of Alabama‟s most critically imperiled fishes and are listed as either S1 or S2 conservation status: E. nuchale (S1), federally endangered; E. boschungi (S2), federally threatened; E. phytophilum (S1), federally endangered; E. ditrema (S1) and E. tuscumbia (S2), state protected; and E. trisella was considered extirpated in Alabama until rediscovered in 2008 (Johnson et al. 2011). The stringent habitat requirements and restricted geographic distributions of these species render them extremely vulnerable to local extirpation and extinction (Etnier 1997). Further, many of the native springs and spring runs occupied by these darters have been capped for industrial and/or residential development, stripped of vegetation, transformed into fishing ponds, or otherwise modified in ways harmful to native species (Mirarchi et al. 2004). Previous genetic studies indicated that the spring endemic E. nuchale consists of three highly structured populations, each of which were recommended as distinct management units in future conservation planning (Mayden et al. 2005; Fluker et al. 2010). Further, Fluker et al. (2010) showed that populations of E. nuchale exhibited low genetic diversity, and thus increased extinction risk, compared to its widespread stream-dwelling relative, E. swaini. Although the genetic characteristics of the federally endangered E. nuchale are now better understood, little is 2 known about the population genetic structure and genetic health, as it relates to conservation practices, for other spring-adapted species throughout the state (Warren et al. 2000; Boschung and Mayden 2004; Mirarchi et al. 2004). Thus, the main objective of this study was to elucidate evolutionary history and population genetic structure and to determine the genetic health of four of Alabama‟s imperiled spring inhabiting darters (E. boschungi, E. ditrema, E. phytophilum, and E. tuscumbia). For each of the four target species, we conducted extensive, range-wide sampling and used a combination of mitochondrial (mt) DNA and nuclear microsatellite (m) DNA data to address the following objectives: 1. Determine population genetic structure within each species. Conservation relevance- Identify appropriate management units for conservation planning and better understand connectivity and migration between populations. 2. Determine levels of genetic variation for distinct populations within each species. Conservation relevance- Populations with low genetic